The present invention relates to novel yeast cells with increased permeability to compounds, such as small organic compounds. In particular, the invention provides genetically modified yeast cells carrying functional, preferably conditionally regulated, copies of HXT9 and HXT11 genes integrated in the chromosome at the PDR1 and PDR3 loci, thereby disrupting the PDR1 and PDR3 gene activity. The invention further relates to methods and compositions for the use of these hyperpermeable yeast cells for screening for compounds that modulate macromolecular interactions. The invention is exemplified by the use of the hyperpermeable yeast cells in such a screening system. In addition, the invention further provides methods of producing the yeast cells of the invention, as well as polynucleotides, vectors, and kits for use of the hyperpermeable yeast cells and the screening methods of the invention.
With recent advances in genome-wide sequencing, studies of protein function and macromolecular interactions have become increasingly important for understanding biological function and for identifying novel therapeutic targets. Screening assays in microbial organisms have been developed to allow rapid identification of genes and gene products involved in various biological activities, including regulation of gene expression, signal transduction, catalysis, and macromolecular interactions important for cellular growth and regulation. For example, a yeast-based screening assay, the so-called yeast two-hybrid screen, has been developed to identify and analyze protein-protein interactions (Fields and Song, 1989, Nature 340:245-246; U.S. Pat. No. 5,468,614). This method allows screening and identification of proteins that specifically interact with a target protein of interest, and has recently been expanded to allow detection of interactions between proteins and RNA (SenGupta et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93: 8496-8501; Wang et al., 1996, Genes Dev. 10: 3028-3040), proteins and nonprotein ligands (Meyerson et al., 1992, EMBO J. 11: 2909-2917), proteins and peptides (Colas et al., 1996, Nature 380: 548-550; Yang et al., 1995, Nucleic Acids Res. 23: 1152-1156), proteins and multiple partners (Osborne et al, 1996, J. Biol. Chem. 271: 29271-29278; Tirode et al., 1997, J. Biol. Chem. 272: 22995-22999), and whole-genome applications (Finley et al., 1994, Proc. Natl. Acad. Sci. USA 91: 12980-12984; Bartel et al., 1996, Nat. Genet 12: 72-77; Fromont-Racine et al., 1997, Nat. Gen. 16: 277-282).
However, less progress has been made at attempts to adapt such yeast screening techniques for use in high-throughput screens for identifying therapeutic drug candidates. A major reason for lack of success in this area is the impermeability of yeast to most organic molecules. To design successful screening techniques in yeast, therefore, the yeast cell membrane must be made accessible to such molecules. Compounds must be able, first, to cross the yeast membrane, and second, once inside the cell, to escape elimination by the yeast detoxification, through metabolic or exocytotic pathways.
Physical and chemical genetic techniques have been used to enhance the permeability of yeast membranes. Permeabilizing agents, such as Polymyxin B sulfate and Polymyxin B nonapeptide, have been used to physically disrupt the integrity of yeast membranes (Boguslawski, 1985, Mol. Gen. Genet. 199:401-405). In addition, yeast genetics and molecular biology techniques have been used to identify genes involved in membrane permeability, and yeast strains have been isolated bearing mutations in such transport pathway genes (e.g., see Brendel, 1976, Mol. Gen. Genet. 147:209-15). A number of yeast genes have been found to be involved in the biosynthesis, maintenance, and degradation of the cell wall and plasma membrane (Lees et al., 1992, ACS SYMP. Ser. 497:246-259; see also, e.g., U.S. Pat. No. 5,821,038), as well as pathways controlling detoxification of organic molecules.
Membrane transport systems are classified into three classes: channels, facilitators, and pumps (Andre, 1995, Yeast 11:1575-1611). Channels are complexes of membrane proteins that mediate passive transport of ions by forming an aqueous diffusion pore. Facilitators mediate the diffusion of solutes across membranes. Many of the facilitators belong to a family, called the Major Facilitator Super-family (MFS), which possess a common structural topology: two 6-transmembrane-spanning helical segments connected by a cytoplasmic loop (Marger and Saier, 1993, Trends Biochem. Sci. 18:13-20). In S. cerevisiae, a network of regulators associated with the phenotype known as pleiotropic drug resistance (PDR), which closely resembles the mammalian MFS, is known to affect cellular transport and drug resistance. Pdr1p and Pdr3p, members of the C6 zinc cluster family of transcriptional regulatory proteins, modulate expression of ABC transporter genes at the transcriptional level (Saunders and Rank, 1982, Can. J. Genet. Cytol. 24:493-502; Katzmann et al., 1994, Mol. Cell. Biol. 14: 4653-4661). Disruption of PDR1 and PDR3, the genes encoding pdr1p and Pdr3p, respectively, results in decreased expression of the ABC transporter PDR5, and thereby increases drug sensitivity of these cells (Nourani et al., 1997, Mol. Cell. Biol. 17:5453-5460). However, expression of two MFS genes from the hexose transporter (HXT) family (Kruckeberg, 1996, Arch. Microbiol. 166:283-292), HXT9 and HXT11, is also regulated by pdr1p and Pdr3p (Nourani et al., 1997, Mol. Cell. Biol. 17:5453-5460). Overexpression of HXT11 in wild-type yeast causes increased drug sensitivity, and, conversely, the loss of either or both of HXT9 and HXT11 expression results in increased drug resistance (Nourani et al., 1997, Mol. Cell. Biol. 17:5453-5460).
Pumps are split amongst two sub-classes, the ATP-Binding Cassette (ABC) transporters and other P-type ATPases, both of which transport solutes against chemical gradients by hydrolysis of ATP. Genes involved in many of these pathways can be modified genetically to alter cellular permeability. Furthermore, it has been reported that the modification of at least two particular genes which control cellular permeability at different levels (i.e., cell wall synthesis or maintenance; plasma membrane synthesis or maintenance; and detoxification or export of endogenous compounds) results in a synergistically increased effect on permeability (see, e.g., U.S. Pat. No. 5,821,038). However, despite such efforts, there is an urgent need to develop new methods for permeabilizing yeast cells for designing high-throughput screening techniques for therapeutic compounds in yeast.
A second major difficulty encountered in attempts at adapting yeast screening technologies into high-throughput screening for candidate therapeutic compounds is the high background of xe2x80x9cfalse positivesxe2x80x9d that typically result from such screens. High backgrounds of non-specific interactions, or xe2x80x9cfalse positivesxe2x80x9d, are a particular problem with yeast two-hybrid screening methods. Currently available methods for screening for molecules that disrupt macromolecular interactions are labor-intensive, and the high backgrounds of non-specific interactions necessitate multiple screening steps. Typically, an initial screen is necessary to identify candidate inhibitors of a target interaction of interest. This screen is then followed by further screening steps to eliminate the false positives. Furthermore, since compounds potentially useful as therapeutics are likely to have relatively weak interactions with their target, identification of drug candidates is even less likely to be successful using this multistep screening procedure.
Therefore, despite great interest and effort in this field, no efficient, sensitive, versatile high-throughput screening system has yet been described for identifying compounds that modulate macromolecular interactions in yeast.
The present invention relates to novel hyperpermeable yeast cells useful for screening for small molecules that modulate macromolecular interactions. The invention is based, in part, on the discovery that the concomitant insertion of functional, preferably conditionally regulated, copies of two yeast genes, involved in hexose transport, into particular genetic loci encoding transcriptional regulators of pleiotropic drug resistance, increases cellular permeability, such as permeability of small molecules. The resultant double insertion/disruption renders the novel cells more permeable to compounds, such as small molecules, than either genetic alteration alone.
The invention provides hyperpermeable yeast cells with increased permeability to compounds such as small molecules. Such hyperpermeable yeast cells are constructed by inserting one or more copies of genes that can increase cellular permeability, and concomitantly disrupting one or more endogenous genes that can decrease cellular permeability. In particular, a hyperpermeable yeast cell comprises a functional HXT9 hexose transporter gene, a functional HXT11 hexose transporter gene; a disrupted PDR1 pleiotropic resistance gene, and a disrupted PDR3 pleiotropic resistance gene, wherein the functional HXT9 gene or the functional HXT11 gene is chromosomally integrated into the disrupted PDR1 gene or the disrupted PDR3 gene.
In one embodiment, the hyperpermeable yeast cell comprises a HXT9 gene chromosomally integrated into the disrupted PDR1 gene, and further comprises an inactivated PDR3 gene and an independently regulated functional HXT11 gene.
In another embodiment, the hyperpermeable yeast cell comprises a HXT9 gene chromosomally integrated into the disrupted PDR3 gene and further comprises an inactivated PDR1 gene and an independently regulated functional HXT11 gene.
In another embodiment, the hyperpermeable yeast cell comprises a HXT11 gene chromosomally integrated into the disrupted PDR1 gene and further comprises an inactivated PDR3 gene and an independently regulated functional HXT9 gene.
In another embodiment, the hyperpermeable yeast cell comprises a HXT11 gene chromosomally integrated into the disrupted PDR3 gene and further comprises an inactivated PDR1 gene and an independently regulated functional HXT9 gene.
In a specific embodiment, the hyperpermeable yeast cell comprises a HXT9 gene chromosomally integrated into the disrupted PDR1 gene, and further comprises a independently regulated functional HXT11 gene chromosomally integrated into a disrupted PDR1 gene.
In another specific embodiment, the hyperpermeable yeast cell comprises a independently regulated functional HXT9 gene chromosomally integrated into the disrupted PDR3 gene, and further comprises a independently regulated functional HXT11 gene chromosomally integrated into a disrupted PDR1 gene.
In yet another specific embodiment, a hyperpermeable yeast cell comprises independently regulated functional HXT9 and HXT11 genes chromosomally integrated into the PDR1 locus, and further comprises a disrupted PDR3 gene.
In yet another specific embodiment, a hyperpermeable yeast cell comprises independently regulated functional HXT9 and HXT11 genes chromosomally integrated into the PDR3 locus, and further comprises a disrupted PDR1 gene.
The invention further provides methods for construction of such yeast cells, comprising inserting HXT9 and HXT11, operably associated with a conditionally regulated promoter into the yeast chromosome at the position of PDR1 and PDR3, respectively, such that the genes PDR1 and PDR3 are disrupted.
The invention further encompasses kits and methods the use of hyperpermeable yeast cells and for screening assays for organic molecules.
The invention is illustrated by a working example provided herein of the engineering of a cell of the yeast S. cerevisiae constructed by inserting HXT9 and HXT11 into the PDR1 and PDR3 loci, respectively. The yeast cells of the invention can be used in any of a number of screening methods, including, for example, enzymatic assays, transcriptional assays, or translational activities, and assays for protein-protein interactions, protein-RNA interactions, protein-nonprotein interactions, protein-peptide interactions, and screening assays for modulators thereof. The invention is further illustrated by an example in which the novel hyperpermeable yeast cells of the invention are used as a host background for a successful yeast two-hybrid dual-bait screening system.
The methods described herein utilize methods and compositions for identifying compounds that modulate macromolecular interactions (e.g., either homotypic or heterotypic protein-protein interactions). For simplicity of description, one protein involved in the protein-protein interaction of interest is referred to herein as a xe2x80x9ctarget proteinxe2x80x9d and a second protein involved in the protein-protein interaction of interest is referred to herein as a xe2x80x9cpartner protein.xe2x80x9d It will be understood that the term xe2x80x9ctarget proteinxe2x80x9d can be considered interchangeable with the term xe2x80x9cpartner proteinxe2x80x9d for the purposes of the methods and compositions described herein. It is also to be understood that the terms can refer to the full-length proteins involved in the protein-protein interactions, or to portions thereof that still exhibit the protein-protein interactions of interest.
As used herein, a xe2x80x9cfunctionalxe2x80x9d copy of a hexose transporter gene, e.g., HXT9 or HXT11, refers to one which can express in either a constitutive, conditionally regulated (e.g., inducible) manner, a polypeptide exhibiting hexose transporter activity, e.g., HXT9 or HXT11 activity.
As used herein, xe2x80x9chyperpermeablexe2x80x9d yeast cells or a xe2x80x9chyperpermeablexe2x80x9d yeast strain, are yeast cells and strains which have an increased sensitivity to one or more test compounds, relative to the sensitivity of the yeast cells and strains without the above-described insertion-disruption of genes that alter cellular permeability. Sensitivity to such compounds and small molecules is determined by measuring the MIC (minimal inhibitory concentration) of such compounds to visible growth of yeast cells, disregarding a haze of barely visible growth of such yeast cells.